Ice rinks are a cultural and recreational cornerstone in Canada, supporting over 800,000 registered skaters, hockey players, and curlers. Maintaining rink surface temperatures between -4 and -8 °C requires energy-intensive refrigeration systems, making these facilities among the largest municipal energy consumers. Statistics indicate that, for each degree increase in surface temperature, approximately 5% of energy consumption can be saved. This study proposes transforming E. coli with modified inaZ genes, native to Pseudomonas syringae, to produce ice-nucleating proteins (INPs) that promote ice formation at higher temperatures. In this study, E. coli will be engineered with two variations of the inaZ gene where the N-terminal domain has been truncated, enabling intracellular protein production. Protein expression of the two variants has been successfully achieved, and ongoing work focuses on increasing protein yield for functional assays. In parallel, molecular dynamics (MD) simulations of INP-water interactions demonstrated the formation of ice lattice structures, with ~600 hydrogen bonds forming over 5 μs. Experimentation on commercial INPs identified a minimum effective nucleation concentration of 0.250 g L⁻¹, capable of inducing ice formation at temperatures as high as -2°C. To evaluate applicability, 3D-printed rink models were developed to simulate rink conditions and assess nucleation efficiency through ongoing functional assays.

This year, our team is continuing our project, but we have made major improvements and important changes. Our project has three main goals:

Objective 1: Purify ice-nucleating proteins (INPs) from genetically modified E.coli bacteria transformed with two different plasmids.

Objective 2: Experimentally compare commercial INPs with our purified ice-nucleating protein product to assess nucleation efficiency through functional assays under ice rink conditions.

Objective 3: Connect with ice industry experts and community rink operators to ensure our project addresses local needs.

In comparison to last year, our team has made several meaningful pivots. In the Wet Lab, we pivoted from a complex Modular Cloning (MoClo) system to traditional gene synthesis and constructed an original INP variant in collaboration with Dr. Elio Cino at the University of Calgary. In the Dry Lab, we made progress in creating computer-modelling simulations of INP variants, and pivoted our experimental approach to testing proteins on 3D-printed model ice rinks, allowing us to simulate freezing conditions accurately. Our Human Practices (HP) team has pivoted our project's application from high-performance arenas to community rinks and has conducted multiple stakeholder interviews to collect statistics, learn about ice maintenance processes, discuss our project's application, and educate our school community about this issue.